Simultaneous Concentration and Real-time Detection of Multiple Classes of Microbial Pathogens from Drinking WaterEPA Grant Number: R833006
Title: Simultaneous Concentration and Real-time Detection of Multiple Classes of Microbial Pathogens from Drinking Water
Investigators: Sobsey, Mark D. , Simmons, Otto D.
Institution: University of North Carolina at Chapel Hill
EPA Project Officer: Klieforth, Barbara I
Project Period: October 18, 2006 through October 17, 2009
Project Amount: $599,999
RFA: Development and Evaluation of Innovative Approaches for the Quantitative Assessment of Pathogens in Drinking Water (2005) RFA Text | Recipients Lists
Research Category: Drinking Water , Water
Develop, collaboratively test, and field evaluate new and improved, rapid ultrafiltration methods and electropositive filter adsorption-elution methods to concentrate viruses (and cellular pathogens such as bacteria and protozoan parasites with the ultrafilter) from waters of different qualities (i.e. particulate and dissolved organic matter). Compare recovery efficiencies to the Environmental Protection Agency’s (EPA) existing 1MDS filter method. Evaluate rapid PEG precipitation for post-filtration sample processing to further concentrate viruses and improved, large volume nucleic extraction methods to remove inhibitors for molecular detection of viral nucleic acids. Optimize viral nucleic acid amplification by real-time RT-PCR for rapid detection of low virus concentrations. Collaboratively (round-robin) test developed methods to validate their performance at selected water virology labs.
Current EPA methods for recovery of viruses use a sole-source, expensive filter with variable performance for some viruses and waters (poor for some enteroviruses and adenoviruses, and for waters with high salinity, particulates and organic matter). We will evaluate two new, improved, and cost-effective filters for virus concentration from water: a disposable, hollow fiber ultrafilter (HFUF) for unified concentration of all microbial pathogen classes and a new, thin-sheet positively-charged filter medium (TSM) fabricated from glass wool prepared in the laboratory and optimized for performance. Properties and preparation methods of the new TSM will be provided to commercial entities for manufacture and mass production. Optimum eluents and elution conditions for a range of enteric viruses will be determined and specified. Subsequent virus concentration and purification from filter eluates and retentates will employ a rapid polyethylene glycol (PEG) precipitation method applied to water concentrates seeded with low levels of a suite of viruses (adenoviruses, enteroviruses and noroviruses) representing those prevent in the USA. Nucleic acid extraction techniques will be optimized for applicability to large sample volumes (1-4 ml) and for effective removal of inhibitors of real-time RT-PCR detection. Real-time RT-PCR will be optimized for detecting target viruses as broad groups as well as select specific viruses (e.g., adenovirus types 40 and 41), with the option to genotype resulting products. The developed methods will be provided to 4 water virology labs recruited for collaborative testing.
Key CCL viruses will be rapidly detected at low levels in water samples concentrated by a rapid HFUF or a new thin-sheet (TSM) electropositive filter adsorption-elution method and compared with the approved EPA method (1MDS VIRADEL). A unified and rapid virus concentration, nucleic acid extraction, and real-time, quantitative RT-PCR amplification technique will be of great value to the water industry because it will provide rapid and sensitive virus recovery and concentration, simplified nucleic acid extraction processes, and robust detection of multiple virus types by rapid real-time PCR and RT-PCR assays.